This application addresses broad Challenge Area (15) "Translational Science" and specific Challenge Topic "15-AR-104 Bone and the Nervous System". Bone adaptation requires osteocytes to detect mechanical signals in situ and integrate the signals in the osteocytic network into appropriate activities in the bone cell system. It is conjectured that a bone cell network mimics a simple neuronal system and can acquire short-term memory to in vivo biomechanical stimuli through the elementary forms of implicit learning, e.g., habituation and sensitization. However, none of these behaviors have been confirmed experimentally in bone cell networks, and little is known about how bone cells behave as a neuronal network. The osteocytic network, recognized as the major mechanical sensor in the bone remodeling process, is the most capable candidate to accommodate the memory function in bone. In this study, we hypothesize that "the ability for elementary forms of plastic change in response to mechanical stimulation is an inherent and fundamental property of the osteocytic network, and the short-term memory in osteocytic networks can be achieved by three elementary forms of implicit learning: habituation, sensitization, and classical conditioning". In the light of our novel in vitro osteocytic network model and experience in calcium signaling research of bone cells, the goals of this study are to: (1) investigate the habituation, sensitization, and classical conditioning behaviors of osteocytic networks to fluid shear stimuli by recording and analyzing the real-time [Ca2+]i wave propagation inside the cell network and to determine the benign and noxious mechanical stimuli in the implicit learning process of osteocytic networks;and (2) investigate the roles of CaMK pathway, gap junction intercellular communication, and P2Y2 receptor in the habituation, sensitization, and classical conditioning of osteocytic networks to mechanical stimuli by using pharmacological inhibitors or siRNA-based knockdown of one of the 3 proteins. If the significant challenges associated with the memory storage in osteocytic network can be overcome, we will have an opportunity to initiate a new paradigm in research of bone mechanotransduction and behavior studies of fundamental physiological systems. The knowledge will profoundly benefit the hunt for strategies and new drugs for the treatment of osteoporosis, which affects millions of people with more than a $10 billion financial burden. This RC1 application proposes to test a novel hypothesis that the implicit memory exists in osteocyte networks under mechanical loading. The real-time intracellular calcium waves in response to various patterns of fluid flow stimulation will be analyzed to study the three elementary forms of implicit learning, i.e., habituation, sensitization, and classical conditioning, in in vitro osteocytic networks. The mechanotransduction mechanisms have a great importance in understanding the etiology of osteoporosis and preventing bone loss in space.